Elucidation of the Cl-transport mechanism of halorthodopsin by time-resolved electric and spectroscopic measurements

首先，我们改进了光伏测量系统，使我们能够检测从1μ秒到500ms的光伏产生。然后，利用时间分辨光伏测量系统，我们研究了盐生盐生杆菌嗜盐菌素的光伏动力学。作为氯离子浓度的函数。光电压由两个主要分量组成，一个具有亚毫秒范围的时间常数，另一个具有不同幅度的毫秒范围时间常数，这两个分量表现出不同的氯离子浓度依赖关系(0.1M到9M)。我们发现，快组分的时间常数与氯离子浓度无关，而慢组分的时间常数在较高的氯离子浓度下呈S形增加。快的和慢的过程分别归因于蛋白质内的电荷(Cl^-)运动和与Cl^-的喷射有关。自发性高血压病膜悬浮液的激光光解研究表明，…它们更多地对应于N中间体的形成和衰变。慢组分的光伏幅值呈现扭曲的钟形氯离子浓度依赖关系，其时间常数的氯离子浓度依赖关系表明在细胞质一侧有一个弱而高度协同的氯离子结合部位(S)(表观K_D约为5M，希尔系数≧为5)。慢过程的光伏幅值和时间常数与氯离子浓度的依赖关系表明，自发弛豫和离子移位之间存在竞争。松弛的时间常数估计为-gt；100ms。还检测了鲨鱼品系卤视紫红质(HR)的电活性。将HR-过量产生的膜组分吸附到聚合物薄膜上后，我们测量了在不同氯离子浓度下光照时的光电电流。光电流的幅度呈钟形的氯离子浓度依赖关系。结果发现，R108Q突变体在高氯离子浓度下也表现出光电流。结果表明，R108对吸收介质中的氯离子很重要，但一旦氯离子位于席夫碱附近，即使没有R108，HR也能表现出生电活性。较少

First of all, we improved the photovoltage measurements system so that it enabled us to examine the photovoltage generation from 1 μsec to 500 msec.Then, using the time-resolved photovoltage measurement system, we examined photovoltage kinetics of halorthodopsin from Halobacterium salinarum. As a function of chloride concentrations. The photovoltage consisted of two major components ; one with a sub-ms range time constant and the other with ms range time constant with different amplitudes., These components exhibited different Cl^- concentration dependencies (0.1 M to 9 M). We found that the time constant for the fast component was relatively independent of the Cl^- concentration, whereas the time constant for the slow component increased sigmoidally at higher Cl^- concentrations. The fast and the slow processes were attributed to charge (Cl^-) movements within the protein and related to Cl^- ejection, respectively. The laser photolysis studies of shR-membrane suspensions revealed that … More they correspond to the formation and the decay of the N intermediate. The photovoltage amplitude of the slow component exhibited a distorted bell-shaped Cl^- concentration dependence and the Cl^- concentration dependence of its time constant suggested a weak and highly cooperative Cl^- binding site(s) at cytoplasmic side (apparent K_D of about 5 M and Hill coefficient ≧ 5). The Cl^- concentration dependence of the photovoltage amplitude and the time constant for the slow process suggested a competition between spontaneous relaxation and ion translocation. The time constant for the relaxation was estimated to be > 100 ms.Electrogenic activity of the halorhodopsin (hR) from shark strain was also examined. After absorbing hR-overproduced membrane fraction onto a thin polymer film, we measured photoelectric current upon illumination at various Cl^- concentrations. The amplitude of the photoelectric current exhibited a bell-shaped Cl^- concentration dependency. It was found that R108Q mutant also exhibited photoelectric current at a high Cl^- concentration. The result indicates that R108 is important for uptaking Cl^- from the medium, but once Cl^- is located near the Schiff base, hR can exhibit electrogenicity even without R108. Less